Recombinant Human Latent TGF-β1

Recombinant Human Latent TGF-β1 is a valuable tool for research applications due to its unique biological properties and experimental versatility. Here are several compelling reasons to use it in your studies:

1. Mimics Physiological TGF-β1 Regulation

Latent TGF-β1 is the predominant form found in vivo, where it is stored in the extracellular matrix and only becomes active upon specific stimuli (e.g., proteolytic cleavage, integrin binding, or mechanical shear). Using the latent form allows you to study the natural activation mechanisms of TGF-β1, which is critical for understanding its role in processes such as wound healing, fibrosis, immune regulation, and development.

2. Controlled Activation in Experimental Models

Recombinant latent TGF-β1 enables researchers to precisely control when and how TGF-β1 is activated. This is particularly useful for:

• Studying activation pathways (e.g., integrin-mediated, protease-mediated, or mechanical activation).
• Investigating the effects of TGF-β1 activation in cell culture or animal models under defined conditions.

3. Longer Plasma Half-Life

Compared to active TGF-β1, the latent form has a longer plasma half-life in vivo, making it suitable for studies requiring sustained exposure or delayed activation of TGF-β1 signaling.

4. Reduced Off-Target Effects

Because latent TGF-β1 is biologically inactive until specifically activated, it minimizes unintended signaling and off-target effects in experimental systems. This is especially important when studying complex biological processes where uncontrolled TGF-β1 activity could confound results.

5. Versatile Applications

Recombinant latent TGF-β1 can be used in a wide range of applications, including:

• Bioassays to study activation mechanisms and downstream signaling.
• Binding assays to investigate interactions with latent TGF-β binding proteins (LTBPs), integrins, or other regulatory molecules.
• Cell culture studies to examine the effects of controlled TGF-β1 activation on cell proliferation, differentiation, migration, and epithelial-mesenchymal transition (EMT).
• Animal models to explore the role of TGF-β1 in disease progression, such as fibrosis, cancer, or autoimmune disorders.

6. Relevance to Disease Mechanisms

Many diseases, including fibrosis, cancer, and autoimmune disorders, involve dysregulated TGF-β1 activation. Using recombinant latent TGF-β1 allows you to model these conditions more accurately and test potential therapeutic interventions that target TGF-β1 activation or signaling.

7. Compatibility with Advanced Techniques

Latent TGF-β1 is compatible with advanced techniques such as:

• Surface plasmon resonance (SPR) to study protein-protein interactions.
• ELISA-based assays to quantify activation or binding.
• Imaging studies to visualize TGF-β1 localization and activation in tissues.

8. Supports Mechanistic Studies

By using recombinant latent TGF-β1, you can dissect the molecular mechanisms underlying TGF-β1 activation and signaling, including the roles of specific proteases, integrins, and extracellular matrix components.

Summary

Recombinant Human Latent TGF-β1 is essential for research that aims to understand the physiological regulation, activation, and biological functions of TGF-β1. Its ability to mimic natural TGF-β1 dynamics, combined with its experimental flexibility and relevance to disease mechanisms, makes it a powerful tool for advancing your research in cell biology, immunology, and disease modeling.

Yes, recombinant human latent TGF-β1 can be used as a standard for quantification and calibration in ELISA assays, though there are important technical considerations to understand.

Standard Composition and Use

Recombinant human latent TGF-β1 standards are specifically designed for ELISA calibration purposes. The latent form consists of the mature TGF-β1 protein non-covalently associated with the latency-associated peptide (LAP), forming the small latent complex (SLC). This recombinant standard is particularly valuable because it accurately represents the native latent TGF-β1 found in biological samples.

Molar Equivalency Considerations

A critical technical point when using latent TGF-β1 standards is that quantification must be based on molar comparison rather than mass comparison. This is because the recombinant LAP homodimer standard differs in molecular weight from the complete latent TGF-β1 complex. Specifically, 1 pM of LAP (54 pg/mL) corresponds to 1 pM of latent TGF-β1 (80 pg/mL). This molar equivalency ensures accurate quantification when measuring native latent TGF-β1 in your samples.

Sample Compatibility

Recombinant latent TGF-β1 standards work effectively with various sample types, including serum, plasma (EDTA, citrate, or heparin anticoagulants), cell culture supernatants, and urine. A significant advantage is that analysis of latent TGF-β1 using these standards does not require pre-treatment of samples to dissociate the latent complex.

Assay Performance

The typical working range for latent TGF-β1 ELISA assays is 0.32-32 pM, with a limit of detection around 0.13 pM. Recovery studies demonstrate excellent accuracy, with typical recovery rates of 95-98% across different sample matrices.

In summary, recombinant human latent TGF-β1 is an appropriate and reliable standard for your ELISA assays, provided you account for the molar equivalency between the LAP standard and the complete latent complex during quantification.

Recombinant Human Latent TGF-β1 has been validated for several key applications in published research, primarily in studies of cell signaling, immunology, fibrosis, and protein interaction assays.

Validated Applications:

• Bioassays: Used to study cellular responses to TGF-β1, including inhibition of cell proliferation, induction of regulatory T cell differentiation, modulation of neutrophil adhesion, and investigation of immune regulatory activities.
• Activation Studies: Employed to investigate mechanisms of latent TGF-β1 activation, including integrin- and protease-mediated activation in cell-based assays.
• Binding Assays: Utilized in surface plasmon resonance and other binding assays to characterize interactions with receptors or binding proteins (e.g., LRRC32, integrins).
• Immunoassay Standards: Used as a standard in ELISA and other immunoassays for quantification and detection of TGF-β1.
• Western Blot Controls: Applied as a control protein in Western blotting to validate antibody specificity and protein detection.
• Media Additive: Added to cell culture media to support protein or antibody production, or to study its effects on cell growth and differentiation.
• In vitro Bioactivity: Validated for assessing bioactivity in cell culture models, including effects on cell proliferation and differentiation.
• Screening Assays: Used in screening and release assays for antibodies or engineered proteins that block or modulate TGF-β signaling.
• Protein Interaction Studies: Used to study protein-protein interactions, such as binding to latent TGF-β binding proteins and other extracellular matrix components.

Representative Published Research Applications:

• Cellular Bioassays: Investigating TGF-β1’s role in regulatory T cell differentiation, neutrophil adhesion, and immune regulation in disease models (e.g., sickle cell disease, rheumatoid arthritis, kidney fibrosis).
• Fibrosis Models: Studying antifibrotic efficacy and safety of TGF-β1 inhibitors in preclinical models of renal fibrosis.
• Protein Activation Mechanisms: Elucidating the structure and activation of latent TGF-β1, including the role of the prodomain in receptor access and activation.
• Cancer Research: Assessing the impact of TGF-β1 on tumor progression and immune cell infiltration in cancer models.
• Matrix Stabilization: Exploring the role of TGF-β1 in cardiac matrix stabilization and vascular rarefaction.

Summary Table of Validated Applications

These applications are supported by peer-reviewed publications and product validation data, demonstrating the versatility of recombinant human latent TGF-β1 in basic and translational research.

To reconstitute and prepare Recombinant Human Latent TGF-β1 protein for cell culture experiments, dissolve the lyophilized protein in sterile water or a mild acidic buffer, then dilute in a protein-stabilizing buffer such as phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) or human serum albumin (HSA) to prevent adsorption and loss of activity.

Step-by-step protocol:

1. Reconstitution:

   • Add sterile distilled water or a mild acidic buffer (e.g., 4 mM HCl) to the lyophilized protein at the volume specified by the manufacturer’s datasheet to achieve the desired stock concentration (commonly 20–100 μg/mL).
   • Gently swirl or invert the vial; avoid vigorous vortexing to prevent protein denaturation.
   • Allow the protein to fully dissolve at room temperature for 10–30 minutes.

2. Stabilization and Dilution:

   • Prepare further dilutions in PBS containing at least 0.1% BSA or HSA to minimize protein adsorption to plasticware and maintain stability.
   • Avoid repeated freeze-thaw cycles by aliquoting the reconstituted protein and storing at –80 °C if not used immediately.

3. Activation (if required):

   • Latent TGF-β1 is inactive and may require activation for biological assays. Activation can be achieved by acidification (e.g., incubating with 1 N HCl for 10 minutes, then neutralizing with 1 N NaOH), heat, or enzymatic treatment, depending on your experimental needs.
   • For cell culture, only activate if your protocol specifically requires active TGF-β1; otherwise, use the latent form as supplied.

Key considerations:

• Latent TGF-β1 is a complex of the mature cytokine with its latency-associated peptide (LAP), rendering it biologically inactive until activated.
• Always consult the specific product datasheet for recommended reconstitution volumes and buffer conditions, as formulations may vary between suppliers.
• Use low-protein binding tubes and pipette tips to minimize loss due to adsorption.

Summary Table:

Always verify the protocol with your specific recombinant protein’s datasheet for optimal results.